Quantitative Structural Assessment of Graded Receptor Agonism

Abstract

Ligand-receptor interactions, which are ubiquitous in physiology, are described by theoretical models of receptor pharmacology. Structural evidence for graded-efficacy receptor conformations predicted by receptor theory has been limited, but is critical to fully validate theoretical models. We applied quantitative structure-function approaches to characterize the effects of structurally similar and structurally diverse agonists on the conformational ensemble of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). For all ligands, agonist efficacy is correlated to a shift in the conformational ensemble equilibrium from a ground state towards an active state, which is detected by NMR spectroscopy but not observed in crystal structures. For the structurally similar ligands, ligand potency is also correlated to efficacy and conformation, indicating ligand residence times among related analogs can influence receptor conformation and function. Our results derived from quantitative graded activityconformation correlations provide new experimental evidence and a platform with which to extend and test theoretical models of receptor pharmacology to more accurately describe and predict ligand-dependent receptor activity.

Structural Mechanism Underlying Ligand Binding and Activation of PPARγ

“…Taken together, the Y473E ligand binding data are consistent with a dynamic activation model (3) whereby PPARγ agonism is associated with ligand-induced stabilization of helix 12, which is dynamic on the µs-ms time scale in apo-PPARγ (18,(23)(24)(25). The NMR data indicate that darglitazone and GW1929 binding to [Y473E]-PPARγ LBD stabilizes the dynamics of most of the orthosteric ligand-binding pocket, likely by binding to the orthosteric pocket in solution though not in the crystallized form.…”

“…Other ligand-bound PPARγ LBD crystal structures obtained from soaking ligand into apo-protein crystals have similarly revealed a ligand bound to this pocket entrance in chain B molecules ( Fig. S4) (24,37,(46)(47)(48)(49)(50)(51), including a crystal structure we previously solved of darglitazone-bound PPARγ LBD (Fig. 4C).…”

“…There is evidence that the functional activity of ligand-bound NRs is associated with a shift in the dynamic LBD conformational ensemble from a ground state to an active state. For example, in the absence of ligand, the PPARγ LBD is conformationally dynamic, samples multiple conformation, and binding of an agonist stabilizes the LBD in an active conformation to a degree correlated with the activity of the ligand (18,(23)(24)(25). The association between ligand-bound NR LBD conformation and graded function is evidence for conformational selection in the mechanism of NR ligand activity (13,15,(19)(20)(21)(22)53).…”

“…Indeed, ligand binding to nuclear receptors is often described to induce an active conformation (4,(11)(12)(13)(14)(15)(16). However, there is evidence from NMR studies on PPARγ that in the absence of ligand the apo-LBD exchanges between transcriptionally active and transcriptionally inactive/repressive conformations (17) suggesting a role for conformational selection in the ligand binding mechanism of NR agonists, which are thought to stabilize an active conformation from a dynamic ensemble of active and inactive/repressive conformations (15,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Taken together, these observations stem from the ability of the ligand-bound NR LBD to exert specific functions such as coactivator interaction and transcription, but not directly on the mechanism of ligand binding to the orthosteric pocket.…”

Structural basis of interdomain communication in PPARγ

“…e molecular and structural basis of ligand-regulated functions of the LBD of nuclear receptors are relatively well understood. For PPARγ, structural biology studies including X-ray crystallography, hydrogendeuterium mass spectrometry (HDX-MS), chemical crosslinking MS (XL-MS), and NMR spectroscopy have revealed how agonist ligands stabilize a transcriptionally active AF-2/helix 12 surface conformation upon binding to the orthosteric ligand-binding pocket in the LBD to promote coactivator protein recruitment and increased expression of PPARγ target genes that drive adipogenesis [2][3][4][5][6] . More recently, we reported a structural mechanism of ligand-dependent corepressorselective PPARγ inverse agonism 7 .…”